The herpes simplex virus (HSV) genome comprises two covalently linked segments, designated long (L) and short (S). Each segment contains a unique sequence flanked by a pair of inverted terminal repeat sequences. The long repeat (RL or RL) and the short repeat (RS or RS) are distinct.
The HSV ICP34.5 (also γ34.5) gene, which has been extensively studied1, 6, 7, 8, has been sequenced in HSV-1 strains F9 and syn17+3 and in HSV-2 strain HG524. One copy of the ICP34.5 gene is located within each of the RL repeat regions. Mutants inactivating both copies of the ICP34.5 gene (i.e. null mutants), e.g. HSV-1 strain 17 mutant 17162 (HSV1716) or the mutants R3616 or R4009 in strain F5, are known to lack neurovirulence, i.e. be avirulent, and have utility as both gene delivery vectors or in the treatment of tumours by oncolysis. HSV-1 strain 17 mutant 1716 has a 759 bp deletion in each copy of the ICP34.5 gene located within the BamHI s restriction fragment of each RL repeat.
ICP34.5 null mutants such as 1716 are, in effect, first-generation oncolytic viruses. Most tumours exhibit individual characteristics and the ability of a broad spectrum first generation oncolytic virus to replicate in or provide an effective treatment for all tumour types is not guaranteed.
HSV 1716 is described in EP 0571410 and WO 92/13943 and has been deposited on 28 Jan. 1992 at the European Collection of Animal Cell Cultures, Vaccine Research and Production Laboratories, Public Health Laboratory Services, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession number V92012803 in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (herein referred to as the ‘Budapest Treaty’).
Squamous cell carcinoma of the head and neck afflicts an estimated 125,000 patients annually in Europe, North America and the Far East. Primary therapy for localized disease is surgery and adjuvant radiotherapy. Tumours recur in approximately one-third of patients. Once the cancer has recurred and/or metastasized, the patient is considered incurable. Combination chemotherapy induces responses in 30-50% of patients but there is no clear impact on survival. There remains an urgent need for more effective therapies12, 13.
There has been much interest in the use of novel therapies in this disease with particular focus on oncolytic viruses by direct intratumoural injection. The use of oncolytic viruses to selectively kill tumours while leaving normal cells unaffected is a very attractive concept as it has the potential to limit the toxicity which occurs with conventional modalities. Recent research has been carried out using intratumoural injections of a selectively replicating adenovirus (Onyx-015) for the local control of recurrent disease. Phase I/II studies involving virus alone and in combination with chemotherapy have produced encouraging results14, 15, 16.
Selectively replicating Herpes simplex viruses HSV may have better efficacy due to its more potent replication and oncolytic potential. HSV171617 is a deletion mutant of herpes simplex virus which fails to synthesise the virulence protein ICP34.5. It has been shown that HSV1716 replicates in actively dividing cells but not in resting or terminally differentiated cells18, 19. In vivo, HSV1716 administration has been carried out in mouse models of a range of cancers including melanoma, teratocarcinoma, glioma, medulloblastoma and mesothelioma. Animals showed improved survival and tumour regression following administration of HSV171620, 21, 22, 23, 24, 25 with no evidence of replication in normal tissue and no toxicity. HSV1716 has been used in Phase 1 trials in patients with glioblastoma multiform (GBM)26, melanoma and head and neck cancer. No toxicity has been experienced and patients who were seropositive pre HSV1716 seroconverted and evidence of virus replication contained within tumours has been obtained.
It has been shown that the novel oncogene SCCRO (Squamous cell carcinoma related oncogene (also called Oncoseq and sometimes called SCRO)) is amplified in 30% of mucosal squamous cell cancers and that overexpression is associated with poor prognosis in head and neck cancer patients.
The Oncoseq nucleic acid sequence was described in U.S. Ser. No. 10/361,725 having publication number US 2004/0009541, published on 15 Jan. 2004. This document is incorporated herein in its entirety by reference. A polynucleotide sequence including an open reading frame of 780 nucleotides for Oncoseq and the amino acid sequence of the 259-residue polypeptide encoded thereby was reported.
US 2004/0009541 describes Oncoseq alleles to be oncogenes identified in primary squamous cell carcinoma tissues as being colocalised with the highest gene duplication peak within the 3q26.3 locus using a positional cloning approach with Oncoseq being highly duplicated in those carcinomas. Overexpression of Oncoseq is described to be correlated with gene duplication, aggressive clinical behaviour and malignant transformation in vitro, making it a strong candidate as the target for 3q amplification. The gene is described to be highly oncogenic and to have a basic region-helix-loop-helix-leucine zipper motif, suggesting it may function as a transcription factor.
RNAi
RNAi utilises small double-stranded RNA molecules (dsRNA) to target messenger RNA (mRNA), the precursor molecule that cells use to translate the genetic code into functional proteins. During the natural process of RNAi, dsRNA is processed into short-interfering RNA (siRNA) duplexes of 21 nucleotides in length, and it is these molecules which recognise and target homologous (endogenous) mRNA sequences for enzymatic degradation (by complementary base-pair binding), resulting in gene silencing.
The advantages of RNAi over other gene-targeting strategies such as anti-sense oligonucleotides include its relative specificity, its enhanced efficacy (only nanomolar quantities of siRNA are required for efficient gene-silencing), and the fact that siRNA treatment feeds into a natural RNAi pathway that is inherent to all cells.
The success of gene-silencing by siRNA can be highly variable depending on the gene target and cell type being targeted.